RE: Histochoice fixative

On Tue, 23 Nov 1999, Penelope Marr wrote:
> My experience with Histochoice was similar to Rob's. The manufacturers
> instructions say to leave tissue in Histochoice for a certain period of
> time. If the tissue is left for longer it autolyses. After my
> experiments I drew the conclusion that Histochoice stabilises certain
> enzymes and antigens but it does not fix the tissue. After processing
> the tissue showed all the symptoms of fixation by whatever reagents it
> was immersed in at the beginning of its processing schedule.
>
> Penny Marr
Penny's observations, written with admirable clarity and brevity,
suggest that this mysterious product is probably not a fixative
but a holding medium such as a solution of a small polyethylene
glycol (PEG 3000 or so) in water, saline or water with about 30%
alcohol (not enough to coagulate most proteins). A secret mixture
of this kind is extolled in Kok & Boon's "Microwave Cookbook" as
a medium in which to suspend specimens for fixation by heating,
delivered by microwave oven.
A recipe for a mixture of this kind is given as "Leyden fixative"
in J.B. Sanderson's excellent book, "Biological Microtechnique"
(Oxford: Bios Scientific Publishers and Roy. Microsc. Soc., 1994).
The idea is that PEG replaces water in the specimen with a
resilient polymer that holds the tissue's macromolecules in
position while they are being cooked or otherwise coagulated.
A doyen of histochemistry, J.E. Scott, coined the catchy phrase,
"fixation by excluded volume," and this was repeated in Pearse's
Histochemistry, which is still very much the Bible of all
histotechnical matters. Consequently the catchy phrase has
been uncritically accepted by some, and with it the idea that
a tissue can be structurally stabilized ("fixed") by replacing
its water molecules with molecules of an unreactive hydropilic
polymer.
This is, of course, rot. Anyone can put a piece of tissue in
"Leyden fixative" and then move it through gently graded solvents
to wax, and will see, as I have done, that the small-scale anatomy
is horribly wrecked. A careful combination of this pseudo-fixation
with exactly the right microwave-delivered heat can probably provide
good preservation of structure at a microanatomical level. Kok &
Boon provide photos that support this assertion. What's probably
happening is that specimens are protected from severe osmotic
damage before being subjected to coagulant fixation that can
otherwise provide great distortion in a specimen bigger than
about 1 cubic mm.
The benefits of coagulant fixation are more easily and reliably
obtained by immersing small specimens in Clarke's or Carnoy's
fluid for a few hours. These fixatives also remove nearly all
the water, so you can go straight into 95% or 100% alcohol,
thereby saving a bit of time.
John A. Kiernan,
Department of Anatomy & Cell Biology,
The University of Western Ontario,
LONDON, Canada N6A 5C1
Phone: (519) 661-2111
FAX (Department): (519) 661-3936
E-mail: kiernan@uwo.ca